A novel approach to the cryopreservation of equine and porcine embryos by vitrification after blastocoelic micromanipulation
Ray, Lucy Eleanor
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Vitrification of equine embryos shows fairly consistent success rates when they are frozen at d 300µm or about 6.5 d after ovulation. In this study, the effects of the reduction of blastocoelic fluid and the microinfusion of a cryoprotectant prior to vitrification were examined on 8 d embryos. The equine embryos used for this project were 805µm (1), 820µm (2), 1120µm (3), 1286µm (4), and 979µm (5). All embryos were graded excellent (1) according to IETS guidelines. The embryos were either assigned to a control group in which no micromanipulation occurred (1-3) or to the experimental group which entailed microinfusion of VS1 (1.4 M glycerol in PBS; 4-5) after aspiration of blastocoelic fluid before microinfusion of VS1 (5). During the following vitrification procedure, the embryos were exposed to VS1 and 2 (1.4M glycerol, 3.6M ethylene glycol in PBS) for 5 minutes, VS3 (3.4M glycerol, 6.6M ethylene glycol in PBS) for 1 minute. The embryo in VS3 was then loaded into a 0.25 ml straw, separated by two air bubbles from columns of 0.5 M galactose. The straws were placed in a cooled plastic goblet surrounded by liquid nitrogen vapors for 1 minute and then finally immersed into the liquid nitrogen. Digital pictures were taken throughout the process. The equine embryos failed to yield a pregnancy with the exception of one (5) which formed an embryonic vesicle at d 15 after ovulation. Ultrasonography at d 28 revealed resorption of the embryo, probably due to heat stress. Due to the low number of equine embryos recovered, porcine embryos were used as a model in another trial. Vitrification of 44 porcine embryos was attempted using identical procedures to those described above. The porcine embryos were cultured for a period of 24 hrs and digital images were also taken. Twenty-six embryos were frozen using the control method and 20 were assigned to the experimental group and treated as described earlier. Initial reexpansion was complete in 16 of the treated and 7 of the control embryos. This showed a significant difference in treatment and control at thaw (p<.0001). All embryos were completely dead at 24 hrs.